Fungal Infection Assay in Zebrafish Embryos

Freshly prepared T. marneffei and A. fumigatus conidia stocks for these experiments were stored at 4 °C for <2 months. For inoculation, 52 hpf tricaine-anesthetized embryos were mounted on an agar mold with head/yolk within the well and tail laid flat on the agar. The fungal conidial suspension was inoculated intramuscularly into a somite aligned to the yolk extension tip for local infection [24 (link),45 (link)] using a standard microinjection apparatus (Pico-Injector Microinjection System; Harvard Apparatus, Holliston, MA, USA) and thin-wall filament borosilicate glass capillary microinjection needle (SDR Clinical Technology, prepared using a P-2000 micropipette puller; Sutter Instruments, Novato, CA, USA). Inoculated embryos were held at 28 °C. The delivered conidial dosage was verified by immediate CFU enumeration on a group of injected embryos [20 (link)]. It took approximately 10 min to commence imaging after inoculation; in this report, the zero time point (t = 0) is taken as the beginning of imaging.